Developmental exposure to bisphenol-A (BPA) at doses within the range of human exposure causes a complex array of adverse effects in animals. These outcomes are also known to be present in human populations and the rise in their occurrence coincides with the massive use of BPA and other endocrine disrupting chemicals in consumer goods. The main hormonal activity of BPA is as an estrogen mimic. Exposure to estrogens throughout a woman's life, including the period of fetal development, is considered a main risk factor for breast cancer. We found that developmental exposure to environmentally relevant doses of BPA altered mammary gland morphogenesis in rodents during the period of exposure and led to the development of pre-neoplastic and neoplastic lesions appearing in adulthood. These results justify adding mammary gland related end points to the large and well-controlled GLP NTP- FDA study. We propose to pursue the following Specific Aims: 1) To test the hypothesis that pre-pubertal mammary gland morphology assessed by morphometries at PND21 is an excellent predictor of pathological outcomes which manifest during adulthood. This aim is based on data obtained independently in our laboratory and that of Dr. S. Fenton (NIEHS). We will compare subjective scoring methods with morphometric ones, and determine which are the features that best predict neoplastic outcomes. We will assess whole mounts of animals exposed until PND21. 2) To test the hypothesis that DNA methylation profiles and concomitant alterations of gene expression in the mammary gland stroma and epithelium at PND21 are predictors of pathological outcomes that manifest during adulthood. 3) To test the hypothesis that perinatal exposure to BPA induces the development of pre-neoplastic and neoplastic lesions. We will assess the development of intraductal hyperplasias, carcinomas in situ and microscopic tumors and the appearance of palpable tumors In animals exposed from I) GD6 until PND 21 and ii) continuously from GD6 to sacrifice. The realization of these Aims will definitively test the hypothesis that perinatal BPA exposure predisposes individuals to mammary cancer and reveal the dose-response pattern. Additionally it will identify candidate molecular markers and morphological signs as predictors of neoplastic outcomes. This knowledge is crucial for the toxicological evaluation of BPA. The success of this project would suggest the addition of some of these mammary gland end points to the toxicological assessment of chemicals.